The present invention relates to a method and an apparatus for concentrating and stabilizing conjugated estrogens from pregnant mare urine onto solid supports in order to obtain a suitable starting material for producing pharmaceuticals that contain the natural mixture of these conjugated estrogens as the active component.
Estrogens are used in the medical field for hormone substitution therapy. In particular, estrogen mixtures are used for the treatment and prophylaxis of change-of-life conditions in women after natural or artificial menopause. In this regard, natural mixtures of conjugated estrogens present in pregnant mares' urine are particularly effective and well tolerated.
The dissolved solid content in pregnant mares' urine (hereinafter abbreviated as “PMU”) can naturally fluctuate in a wide range, generally 40-90 g total solids per liter. In addition to urea and other typical components of urine, phenolic substituents in quantities of approximately 2-5 wt-% relative to total solids are contained in PMU solids. These phenolic substituents include cresols and dihydro-3,4-bis[3-hydroxyphenol)methyl]-2 (3H)-furanone, known as HPMF. These substances may be present in the free or conjugated form. PMU contains a natural mixture of estrogens which are very prevalent in the conjugated form, as the sulfuric acid hemiester sodium salt, for example, (hereinafter abbreviated as “sulfate salt”). The content of conjugated estrogens (hereinafter abbreviated as “CE”) can be calculated as estrogen sulfate salt, and ranges between 0.3 and 1 wt-%, relative to total solids.
Various methods have been described in the prior art for the direct preparation and recovery of conjugated estrogens contained in PMU. Extracts containing conjugated estrogens are usually obtained from PMU by extraction with a polar organic solvent immiscible or sparingly miscible in water, such as acetic acid ethyl ester, n-butanol, or cyclohexanol, for example. However, numerous problems arise in such liquid-liquid extractions, such as intense foam formation, sedimentation, emulsification, and strong phase separation. Several extraction steps are generally required, which results in losses and only partial recovery of the estrogen. To avoid these disadvantages, therefore, a number of solid phase extraction methods have been proposed in the prior art.
In order to obtain small quantities of urine and plasma liquids for analytical determination of estrogens by gas chromatography, Heikkinnen et al. (Clin. Chem. 27/7, (1981), 1186-1189) and Shackleton et al. (Clinica Chimica Acta 107 (1980) 231-243) have described solid phase extraction of estrogens using a cartridge comprising silanized silica gel containing octadecylsilane groups (Sep-Pak® C18 cartridge, manufactured by Waters Ass. Inc., Milford, Mass., USA). In this method the estrogens were eluted from the cartridge with methanol.
In 1968, H. L. Bradlow (see Steroids 11 (1968), 265-272) proposed the use of Amberlite XAD-2™, a neutral, nonpolar hydrophobic polystyrene resin from Rohm and Haas for the extraction of conjugated estrogens. The stated adsorption capacity is low. According to Bradlow, optionally diluted urine was conducted at a low throughput rate through a column containing the resin. The estrogens were eluted with methanol or ethanol.
Recent patent applications describe methods for obtaining an extract containing a natural mixture of conjugated estrogens from mare urine by solid phase extraction of the mixture of conjugated estrogens from pregnant mare urine on, for example, RP silica gel (International Patent WO 98/08525) or on nonionized, semipolar polymeric adsorbent resins (International Patent WO 98/08526).
In addition to the aforementioned optimization of the direct, complete preparation of pregnant mare urine (PMU) for obtaining natural mixtures of CE, steps preceding the preparation, such as securing protective storage for the estrogens and handling the collected urine at the collection site, are of particular importance for the yield and quality of the estrogen-containing raw materials and of the natural mixture of conjugated estrogens isolated therefrom. In this regard, measures to optimize the transport of the collected estrogen-containing raw materials from the collection site to the site of actual preparation and isolation of conjugated estrogens are also desirable.
The complete processing of PMU, for example by solid phase extraction, requires qualified personnel trained in chemical and pharmacological methods in order to observe under well-controlled conditions the exacting requirements for purity and quality of a substance or substance mixture used as a pharmaceutical agent. Collection of the urine (PMU), however, usually takes place in the normal environment of the pregnant mares, that is, at stud farms in rural and often remote locations. As a rule, only a simple infrastructure is present here, and the collection and handling of urine (PMU) is typically carried out by ordinary personnel following instructions, so that high standards for handling the collected urine cannot be expected.
However, conjugated estrogens in the composition excreted in pregnant mare urine are a complex mixture containing in particular sodium estrone sulfate, sodium equilin sulfate, and other CE. It is very important in the isolation of CE that the PMU be processed as quickly as possible in fresh condition. Under extended storage urine rapidly begins to decompose, turning a dark color and giving off the odor of ammonia, with the CE content declining drastically. The rate of decomposition is a function of the storage conditions and the purity criteria. For this reason, the collected urine (PMU), which involves large volumes of liquid, hitherto has had to be transported daily from the collection site to a centralized processing point to assure the most rapid processing possible.
Hence, there is a pressing need for suitable methods and apparatus for concentrating and stabilizing conjugated estrogens contained in pregnant mare urine directly at the collection site, in a manner that is most protective of the product and allows the greatest ease in handling, and for the most efficient transport from the collection site to the site of processing and isolation.